Manuscripts 

Cancer is the most common cause of death in adult canines. Similar to humans, canines have a one-third chance of developing cancer in their lifetime. However, currently, there are no established screening methods for cancer early detection in canines. We developed a shallow whole-genome sequencing (sWGS)-based liquid biopsy test called PetCanSeek to capture two common genomiclepigenetic features of cancer,copy-number aberration (CNA) and fragment size (FS), for multi-cancer early detection in canines.

Canine cancer research is a complex subject involving many subsidiary subject areas. In this field, multiple disciplines such as biomedicine, genetics, immunology, and oncology are interwoven to deeply explore and understand the pathogenesis, prevention strategies, and treatment methods of canine cancer. Canine genomics perceives canine cancer as "a disease of the genome" and categorizes genomic alterations in canine cancer. Liquid biopsy has been described as "the next frontier in veterinary cancer care", establishing an emerging technology focused on cell-free DNA methylation profiling, cell-free DNA fragmentomics analysis, shallow whole-genome sequencing, and multi-omics analysis. In humans, given its non-invasive advantages, the utilization of liquid biopsy testing has already become integral to every step of the clinical journey of cancer screening, auxiliary diagnosis, minimal residual disease detection, therapeutic response monitoring, and recurrence monitoring. In principle, veterinary use of this technology has a similar scope of applications. The transfer of this technology from human medicine into veterinary medicine will facilitate its swift adoption, for the benefit of these veterinary patients.

Objective: Cancer is currently the most common cause of death in adult dogs. Like humans, dogs also have a one-third chance of developing cancer in their lifetime. We used shallow whole-genome sequencing (sWGS) to analyze blood cell-free DNA (cfDNA) from four tumor-bearing dogs (one with benign and three with malignant tumors) and 38 healthy dogs.

Results: Similar to the results observed in healthy dogs, no copy number aberration (CNA) was detected in the dog with benign lipomas, and the distribution of cfDNA fragment size (FS) closely resembled that of healthy dogs. However, in the three dogs diagnosed with malignant tumors, each dog exhibited varying degrees and quantities of CNAs. Compared to the distribution of FS in healthy dogs, cancer dogs exhibited a noticeable shift towards shorter lengths. These findings indicated that CNA and FS values derived from sWGS genomic profiling can be used for non-invasive cancer detection in dogs.

In population-wide cancer screening, three key issues need to be focused on: the number of cancer cases identified, the number of false positives, and the cost. OncoSeek is a multi-cancer early detection (MCED) test using seven protein tumor markers and artificial intelligence. SeekInCare is an MCED test that integrates the seven protein tumor markers and four cancer genomic features from cell-free DNA by shallow whole-genome sequencing. In a two-step approach, the initial screening is conducted using OncoSeek, and SeekInCare is then used as the secondary test for individuals who tested positive by OncoSeek. We simulated a screening in five million adults ages ≥50 years with a cancer incidence rate of 1.9%. Whereas at 91.0% specificity OncoSeek had 441,450 false positives, using the two-step approach significantly reduced false positives to 34,335 (0.7%). Although SeekInCare and Galleri identified more cancer cases (32,015 and 27,455, respectively) than the two-step MCED (21,280), their total costs reached $3,750 million and $4,745 million, respectively. As the positive predictive value of two-step MCED (38.3%) was comparable with SeekInCare (27.7%) and Galleri (38.3%), it reduced the cost by 5.3-fold and 6.6-fold, respectively, amounting to a total cost of $713.6 million and a cost of $143 per individual screened. The cost of per cancer case detected was $117,133 for SeekInCare and $172,828 for Galleri, which were 3.5-fold and 5.2-fold higher, respectively, than the two-step MCED ($33,534). The two-step approach not only significantly reduces false positives but also cuts down the screening cost substantially, making it a cost-effective strategy for population-wide cancer screening.